This invention relates to artificial chromosome constructs containing foreign nucleic acid sequences, such as viral nucleic acid sequences, and methods of using these constructs for therapy and for recombinant virus production.
Artificial chromosomes are large, DNA-based vectors that have been used extensively in the construction of DNA libraries for complex genome mapping and analysis. Artificial chromosomes have been derived from yeast (yeast artificial chromosomes: YACs), bacteria (bacterial artificial chromosomes: BACs, and P1-derived artificial chromosomes: PACs), and mammals (mammalian artificial chromosomes: MACs), such as humans (human artificial chromosomes: HACs). These vectors include elements derived from chromosomes that are responsible for replication and maintenance, and are capable of stably maintaining large genomic DNA fragments.
Herpes Simplex virus (HSV) is the prototypic human herpes virus. Despite the fact that HSV is a human pathogen, there has been a great deal of interest in using HSV as a therapeutic agent. The HSV genome has been sequenced, and many HSV mutants have been generated and used specifically in this context. Generation of HSV mutants has been carried out by using drug selection or by co-transfection of cells with plasmid DNA, usually modified by insertion of a marker gene, and intact viral DNA. Mutants are identified by screening for either drug resistance or recombination and expression of the marker gene, or by plaque hybridization. Another method that has been used to generate herpes virus mutants involves the use of cosmid sets that, taken together, contain complete herpes virus genomes. For example, cosmid sets that contain the entire genomes of Pseudorabies virus (PRV), Varicella-Zoster virus (VZV), Herpes Simplex virus (HSV), Cytomegalovirus (CMV), and Epstein-Barr virus (EBV) have been created. In constructing complete viral genomes from these cosmids, viral sequences are released from the cosmid backbones and transfected into cells. Viral plaques are produced via recombination between the overlapping fragments, which together represent the entire genome. Specific mutations are made in the viral genomes by manipulating the cosmid DNA.
The invention provides artificial chromosome constructs containing foreign nucleic acid sequences, such as viral nucleic acid sequences, and methods of using these artificial chromosome constructs for therapy (e.g., gene therapy) and recombinant virus production.
Accordingly, in one aspect, the invention features an artificial chromosome construct containing a nucleic acid sequence that directs formation of a recombinant virus (e.g., a lytic or a non-lytic virus) upon introduction into a cell. Optionally, the artificial chromosome construct, either in the artificial chromosome portion or in the nucleic acid sequence portion, further includes a heterologous nucleic acid sequence that, for example, encodes a therapeutic gene product, such as a growth factor, a hormone, an enzyme, a vaccine antigen, a cytotoxin, an immunomodulatory protein, an antisense RNA molecule, or a ribozyme. The artificial chromosome portion of the construct can be derived from a bacterial artificial chromosome, a P1-derived artificial chromosome, a yeast artificial chromosome, or a mammalian (e.g., human) artificial chromosome. The recombinant virus encoded by the nucleic acid sequence included in the artificial chromosome construct can be a herpes virus, such as a herpes simplex virus. Other viruses that can be encoded by the nucleic acid sequence are listed below.
In another aspect, the invention features a method of producing a recombinant virus in a cell, for example, a cell in a mammal, in which an artificial chromosome construct as described above is introduced into the cell. This method can further involve introducing into the cell an amplicon that is packaged into a recombinant virion upon introduction of the artificial chromosome construct into the cell.
The invention also provides a method of introducing a heterologous nucleic acid sequence into a cell, for example, a cell in a mammal, in which an artificial chromosome construct as described above is introduced into the cell.
Also included in the invention is a method of killing a cell, for example, a cell in a mammal (e.g., a cancer cell), in which an artificial chromosome construct as described above is introduced into the cell.
The invention also features a cell having an artificial chromosome construct stably integrated into its genome. In one example, the artificial chromosome construct includes a nucleic acid sequence that encodes an HSV genome in which an immediate early gene is mutated or deleted (see below). The invention also includes methods of making these cells.
The invention provides many advantages. For example, when bacterial artificial chromosome constructs are employed in the invention, the constructs can be easily and efficiently propagated in bacteria. This is particularly advantageous in the case of constructs including viral genes that would be cytopathic if the constructs were propagated in mammalian cells. This is also advantageous in the manufacturing of recombinant viruses, because large-scale, bacterial culture methods can be used, rather than methods employing mammalian cell culture. An additional advantage of the invention is that, in contrast to cosmid-based systems for virus production (see above), which rely on recombination of several molecules to reconstitute a complete viral genome, an entire viral genome can be contained in a single artificial chromosome construct, providing increased efficiency and genetic stability. As is discussed further below, this is particularly advantageous in the trans packaging of amplicons into recombinant viruses using the methods of the invention.